G protein mediated signal transduction pathways are involved in the responses of organisms and their constituent cells to a wide variety of stimuli including light, gustants, odorants, hormones, and neurotransmitters. G protein-mediated signal transduction occurs when an agonist binds selectively to its heptahelical receptor leading to the activation of a heterotrimeric G protein. These G proteins are composed of alpha (Ga), beta (Gb) and gamma (Gg) subunits, and when activated they are able to regulate the activity of specific effector proteins. Most cells harbor multiple G protein signaling pathways with the potential to work at cross purposes unless they are appropriately segregated from one another. Mounting evidence suggests that this is achieved by assembling receptors, G proteins and effectors into signaling complexes. Several different fluorescence based techniques are being used to investigate when, where and under what circumstances signaling complexes are formed and dissolved in living cells. These techniques, known as resonance energy transfer (RET), and bimolecular fluorescence complementation (BiFC), can provide both spatial and temporal information about protein complexes. RET involves the exogenous expression of fusion proteins tagged with either luciferase (Luc) or a fluorescent moiety. The fluorescent moiety can be a fluorescent protein, such as green (GFP) or yellow fluorescent protein (YFP), or a tetracysteine motif CCPGCC that is capable of binding biarsenical derivatives of fluorescent compound (ie. FlAsH). RET occurs when the energy from Luc or a fluorescent tag (the donor) on one protein is transferred to the fluorescent tag (the acceptor) on another protein causing the acceptor to fluoresce. This only occurs if the donor and acceptor tags are juxtaposed (less than 100 angstroms apart) because the proteins they are fused to associated to form a complex. BiFC is based on the fact that the complementary N- and C-terminal fragments of YFP (YN and YC, respectively) are not themselves fluorescent, but will reconstitute a fluorescent YFP molecule if they are brought together by being fused to proteins that associate to form a complex.[unreadable] [unreadable] The D4.2 dopamine receptor (D4.2R) inhibits the effector protein adenylyl cyclase (AC) by activating the inhibitory heterotrimeric G protein, Gi. Fusion proteins of D4.2R with Luc or a fluorescent protein are inactive. To create a fluorescent D4.2R that could be used in RET experiments a CCPGCC motif was added to the C-terminus (D4.2R-PGCC) or at two different positions within the third intracellular loop (D4.2R-G259C and D4.2R-G275C). The tetracysteine motif did not affect cell surface expression, ligand binding to the receptor, or agonist mediated-inhibition of AC, and FlAsH binding to this motif produced a fluorescent D4.2R that could be used as an acceptor for RET experiments. RET occurs when either D4.2R-G257C or D4.2R-G275C was co-expressed in HEK 293 cells with a Luc tagged AC (AC-Luc). There was no significant RET between AC-Luc and D4.2R PGCC. RET also occurred between the tagged D4.2R and Luc-tagged Gg. These data suggest that both G protein and AC are part of a signaling complex with D4.2R.[unreadable] [unreadable] The beta2-adrenergic receptor (b2AR) stimulates AC by activating the stimulatory heterotrimeric G protein, Gs. RET was observed when the b2AR was tagged with Luc (b2AR-Luc) and co-expressed with D4.2R-G259C or D4.2R-G275C suggesting that both stimulator and inhibitory receptors involved in the dual regulation of AC are present in the same signaling complex. There was no significant RET between b2AR-Luc and D4.2R-PGCC even though it was functionally indistinguishable from wild type D4.2R. This is likely a consequence of the donor and acceptor tags being to too far apart or in the wrong orientation for RET to occur. RET between Luc-tagged signaling proteins and CCPGCC-tagged D4.2R occurred in the absence of signaling, and was not affected by agonist-mediated signaling.[unreadable] [unreadable] BiFC was combined with RET to demonstrate the simultaneous presence of three different protein in a signaling complex. BiFC occurred when b2AR tagged with YC (b2AR-YC) and Gg tagged with YN (YN-Gg) were co-expressed in HEK 293 cells. RET occurred when AC-Luc was co-expressed with b2AR-YC and YN-Gg indicating that receptor, G protein and effector are part of the same signaling complex. Experimental evidence also supports the hypothesis that G protein-mediated signaling complexes are formed before they reach the plasma membrane. RET together with subcellular fractionation demonstrated that a complex of AC and the b2AR are present on intracellular membranes. Further, dominant-negative (DN) GTPases (Rab1 and Sar1) which block anterograde trafficking out of the endoplasmic reticulum (ER) have no effect on either b2AR/AC, Gg/AC or b2AR/Gg interactions. However, DN Rab1 and Sar1 constructs (but not DN Rabs 2, 6, 8 or 11) prevent the inclusion of Ga subunits in AC signaling complexes suggesting Ga becomes part of the complex at some point beyond the ER. In summary our data support the hypothesis that heptahelical receptors, G proteins and effectors are assembled into complexes before being transported to their target membrane, and that these complexes persist when the signal transduction pathway is activated by an agonist. This arrangement helps to explain the specificity and efficacy that is often observed during G protein-mediated signal transduction.